水牛睾丸特异LDHC基因的原核表达及生物学活性研究

Prokaryotic Expression and Biological Activity Analysis of Buffalo Testicles LDH-C Gene

目的:克隆水牛睾丸特异Ldhc基因全长在大肠杆菌中原核表达,研究其生物学活性。方法:以水牛睾丸组织为材料提取总RNA,RT-PCR扩增Ldhc cDNA,PCR获得水牛全长Ldhc基因;连接pET-32b构建表达质粒pET-32b-Ldhc;转化BL21(DE3)大肠杆菌,IPTG诱导表达并利用SDS-PAGE分析。体外Ni离子柱纯化目的蛋白,Western印迹鉴定其抗体结合活性,同工酶谱鉴定其生物学活性。结果:成功制备了表达质粒pET-32b-Ldhc;IPTG诱导得到56kDa目的蛋白;Ni柱纯化获得纯度90%以上的蛋白;Western印迹显示目的蛋白具有特异的抗体结合活性;同工酶活性染色证明其具有乳酸脱氢酶活性。结论:试验成功制备了水牛睾丸LDH-C4蛋白,并初步验证了其生物学活性,为我们进一步探讨LDH-C4的功能及免疫节育疫苗的制备等奠定了基础。

Objective： Clone and Prokaryotic expression of buffalo testicles LDH-C4,Study its biological activity.Method： Total RNA was extracted from buffalo testes and was reverse transcribed RT-PCR.The full length ORF of cattle Ldhc was amplified by PCR.Ldhc gene linked with expression vector pET-32b to construct the recombinant expression plasmid pET-32b-Ldhc,and then transformed into the E.coli BL21（DE3） competent cells.The expression was induced by IPTG,and the products were analyzed by SDS-PAGE.Ni-NTA His Bind Resin affinity chromatography was used to obtain purified LDH-C4 proteins.The antibody bond ability was tested by Western blot assay.The enzyme activity of LDH-C4 was detected by isozymogram.Result：Prepared the expression plasmid pET-32b-Ldhc successful.The molecular weight of the expression products were approximately 56kDa,Purity of the LDH-C4 proteins90% after purified by Ni-NTA and renaturation,Western blot showed that the recombinant protein with a specific antibody binding activity,and the isozymogram demonstrated its biologic activity as lactate dehydrogenase.Conclusion：These results showed that we successfully prepared the testicular LDH-C4 protein of buffalo,and validated its biological activity preliminary,which will establish a basis for further developing the function of LDH-C4 and preparing immunization vaccine.